Genome and transcriptome characterization of the glycoengineered Nicotiana benthamiana line ΔXT/FT

Abstract

Background The allotetraploid tobacco species Nicotiana benthamiana native to Australia has become a popular host for recombinant protein production. Although its usage grows every year, little is known on this plant’s genomic and transcriptomic features. Most N. benthamiana accessions currently used in research lack proper documentation of their breeding history and provenance. One of these, the glycoengineered N. benthamiana line ΔXT/FT is increasingly used for the production of biopharmaceutical proteins. Results Based on an existing draft assembly of the N. benthamiana genome we predict 50,516 protein –encoding genes (62,216 transcripts) supported by expression data derived from 2.35 billion mRNA-seq reads. Using single-copy core genes we show high completeness of the predicted gene set. We functionally annotate more than two thirds of the gene set through sequence homology to genes from other Nicotiana species. We demonstrate that the expression profiles from leaf tissue of ΔXT/FT and its wild type progenitor only show minimal differences. We identify the transgene insertion sites in ΔXT/FT and show that one of the transgenes was inserted inside another predicted gene that most likely lost its function upon insertion. Based on publicly available mRNA-seq data, we confirm that the N. benthamiana accessions used by different research institutions most likely derive from a single source. Conclusions This work provides gene annotation of the N. benthamiana genome, a genomic and transcriptomic characterization of a transgenic N. benthamiana line in comparison to its wild-type progenitor, and sheds light onto the relatedness of N. benthamiana accessions that are used in laboratories around the world. Electronic supplementary material The online version of this article (10.1186/s12864-019-5960-2) contains supplementary material, which is available to authorized users.

Prolyl Hydroxylase Paralogs in Nicotiana benthamiana Show High Similarity With Regard to Substrate Specificity

“…Interestingly, aligning several biological replicates of amplified and cloned sequences of Nb-P4H4 and Nb-P4H10 with the sequences of the N. benthamiana database showed some discrepancies in sequence identities ( Supplementary Table 4 ). This potentially represents natural differences in N. benthamiana accessions ( Schiavinato et al, 2019 ). However, this did not disrupt enzyme activity probably attributed to the fact that the amino acids in key positions for binding cofactors and substrate were unaffected by these mutations.…”

Identification of two subtilisin‐like serine proteases engaged in the degradation of recombinant proteins in Nicotiana benthamiana

“…these SBTs were cloned from reverse-transcribed N. benthamiana RNA and found to closely match those predicted from the most current N. benthamiana gene assemblies [27,28].…”

Plastid genomics ofNicotiana(Solanaceae): insights into molecular evolution, positive selection and the origin of the maternal genome of Aztec tobacco (Nicotiana rustica)

“…Some members of Nicotiana offer several research advantages, including extensive phenotypic diversity, amenability to controlled hybridizations and ploidy manipulations, high fecundity, and excellent response to tissue culture ( Lewis, 2011 ). Consequently, N. tabacum and N. benthamiana Domin have become model organisms in the generation of new knowledge related to hybridization, cytogenetics, and polyploid evolution ( Goodin et al, 2008 ; Zhang et al, 2011 ; Bally et al, 2018 ; Schiavinato et al, 2019 ). The first complete plastome nucleotide sequence was published in 1986 for N. tabacum ( Shinozaki et al, 1986 ).…”